Identification of Wee1 As a Novel Therapeutic Target for Mutant RAS

ORIGINAL ARTICLE Identiﬁcation of Wee1 as a novel therapeutic target for mutant RAS-driven acute leukemia and other malignancies

E Weisberg1,4, A Nonami1,4, Z Chen1, F Liu2, J Zhang3, M Sattler1, E Nelson1, K Cowens1, AL Christie1, C Mitsiades1, K-K Wong1, Q Liu2, N Gray3 and JD Grifﬁn1

Direct targeting of rat sarcoma (RAS), which is frequently mutated, has proven to be challenging, and inhibition of individual downstream RAS mediators has resulted in limited clinical efficacy. We designed a chemical screen to identify compounds capable of potentiating mammalian target of rapamycin (mTOR) inhibition in mutant RAS-positive leukemia, and identified a Wee1 inhibitor. Synergy was observed in both mutant neuroblastoma RAS viral oncogene homolog (NRAS)- and mutant kirsten RAS viral oncogene homolog (KRAS)-positive acute myelogenous leukemia (AML) cell lines and primary patient samples. The observed synergy enhanced dephosphorylation of AKT, 4E-binding protein 1 and s6 kinase, and correlated with increased apoptosis. The specificity of Wee1 as the target of MK-1775 was validated by Wee1 knockdown, as well as partial reversal of drug combination-induced apoptosis by a cyclin-dependent kinase 1 (CDK1) inhibitor. Importantly, we also extended our findings to other mutant RAS- expressing malignancies, including mutant NRAS-positive melanoma, and mutant KRAS-positive colorectal cancer, pancreatic cancer and lung cancer. We observed favorable responses with combined Wee1/mTOR inhibition in human cancer cell lines from multiple malignancies, and inhibition of tumor growth in in vivo models of mutant KRAS lung cancer and leukemia. The present study introduces for the first time Wee1 inhibition combined with mTOR inhibition as a novel therapeutic strategy for the selective treatment of mutant RAS-positive leukemia and other mutant RAS-expressing malignancies.

Leukemia (2015) 29, 27–37; doi:10.1038/leu.2014.149

INTRODUCTION developing individual targeted therapies a challenge. There is Point mutations in the rat sarcoma (RAS) family, which is thus potential clinical benefit to be gained from the use of a multi- comprised of neuroblastoma RAS viral oncogene homolog (NRAS), targeted therapy approach as opposed to a more narrowly kirsten RAS viral oncogene homolog (KRAS) and V-Ha-ras Harvey focused strategy to enhance efficacy and circumvent the RAS viral oncogene homolog, are the most frequently occurring emergence of drug resistance. mutation in human proto-oncogenes, with around 20–30% of all One promising approach to improving the clinical efficacy of human malignancies characterized by constitutively activated inhibitors of RAS-mediated transformation is the identification of RAS.1 The incidence of mutations in NRAS and KRAS in acute combinations of drugs that target either multiple RAS-driven myelogenous leukemia (AML) has been reported to be pathways, or block bypass or resistance pathways. As mTOR is an approximately 10–11% and 5%, respectively, and around 12% established and important downstream effector of RAS in for myelodysplastic syndromes.2,3 The incidence of NRAS leukemia and mTOR inhibitors are under clinical investigation mutations has been reported to be approximately 10% in for the treatment of mutant RAS-associated malignancies childhood acute lymphoblastic leukemia (ALL).4 (reviewed in McCubrey et al.7), we developed a chemical screen Despite its prevalence and significance with respect to to identify agents capable of enhancing the activity of an mTOR transformation, direct molecular inhibition of mutant RAS has inhibitor, Torin 1.8 As anticipated, this screen identified inhibitors thus far been difficult because of its biochemistry and structure, of phosphatidylinositol 3-kinase (PI3K) and MAPK (mitogen- although KRAS (G12C) mutant-specific inhibitors, which depend activated protein kinase)/ERK kinase (MEK), but unexpectedly on mutant cysteine for their selective inactivation of this mutant, also identified MK-1775, an inhibitor of Wee1, which has not have recently been reported and are in early stages of previously been linked to RAS signaling. development.5,6 The potential to interrupt mutant RAS signaling Wee1 is a protein kinase and inhibitory regulator of the G2/M by inhibiting the major signaling pathways of RAS, PI3K/PTEN checkpoint that prevents cells from going through mitosis by (phosphatase and tensin homolog)/AKT/mammalian target of inhibiting the activity of cell division cycle 2 (cdc2; also known as rapamycin (mTOR) and Raf/MEK/ERK (extracellular signal- cyclin-dependent kinase 1 (Cdk1)).9–11 In response to DNA regulated kinases), has prompted the development of selective damage, such as that induced in tumor cells by cytotoxic inhibitors of these pathways. However, the redundant nature and agents, Wee1 inactivates cdc2 through phosphorylation of its cross-talk of the highly complex signaling cascades associated Tyr15 residue, thus leading to G2 arrest; this allows transformed with RAS can lead to drug resistance and therefore make cells the time needed for repair of damaged DNA and thus confers

1Department of Medical Oncology, Dana Farber Cancer Institute, Harvard Medical School, Boston, MA, USA; 2High Magnetic Field laboratory, Chinese Academy of Sciences, Hefei, People’s Republic of China and 3Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA. Correspondence: Dr E Weisberg or Professor JD Griffin, Department of Medical Oncology, Dana-FarberCancerInstitute,450Brookline Avenue, Mailstop Mayer 540, Boston, MA 022150, USA. E-mail: [email protected] or james_griffi[email protected] 4These authors contributed equally to this work. Received 15 January 2014; revised 21 April 2014; accepted 28 April 2014; accepted article preview online 5 May 2014; advance online publication, 6 June 2014 Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 28 a survival advantage.9–11 Inhibition of Wee1 by MK-1775 results in AML patient cells the removal of cdc2 Tyr15 phosphorylation, activation of cdc2 and Mononuclear cells were isolated from samples from AML patients the entrance of cells into the mitotic phase of the cell cycle.12 identified as harboring mutant NRAS. Cells were tested in liquid culture Wee1 inhibition has been thought to potentiate the effects of (Dulbecco’s modified Eagle’s medium, supplemented with 20% fetal chemotherapy by causing transformed cells with damaged DNA bovine serum) in the presence of different concentrations of single and to go through an untimely mitosis and undergo apoptosis.13 combined agents. All blood and bone marrow samples from AML patients In fact, MK-1775 has been shown to enhance the activity of were obtained under approval of the Dana Farber Cancer Institute 14,15 Institutional Review Board. chemotherapy agents against a variety of malignancies. Mutant NRAS-positive AML5 cells were derived from a 46-year-old patient However, little has been reported on the ability of MK-1775 to with AML, with maturation, harboring NRAS-G13D, with 49% bone marrow potentiate the effects of targeted and selective small-molecule blasts. Mutant NRAS-positive AML6 cells were derived from a 69–year-old inhibitors that offer the potential benefit of less adverse effects patient with AML, harboring NRAS-G13D, with 43% bone marrow blasts. and better tolerability as compared with standard cytotoxic agents. The present study is the first report of the ability of Knockdown of Wee1 by small hairpin RNA (shRNA) Wee1 inhibition to increase the effectiveness of targeted pLKO.1puro lentiviral shRNA vector particles against Wee1 were purchased inhibition of RAS signaling against mutant RAS-positive AML. We from Sigma-Aldrich (St Louis, MO, USA). Cells were incubated with the viral also demonstrate the ability of MK-1775 to potentiate mTOR particles in the presence of 8 mg/ml polybrene for 24 h, and the cells were inhibition in the broader context of additional mutant RAS- selected with 1–2 mg/ml puromycin for 72 h. Following selection, cells were expressing malignancies, including ALL, melanoma, colorectal used for the studies described. cancer, pancreatic cancer and lung cancer. The sequence of shRNA are follows: GFP: 50-ACAACAGCCACAACGTCTATA-30 Wee1: 50-CTAGAAAGAGTGCAGAACAAT-30. MATERIALS AND METHODS Mouse studies Large scale production of perturbagen-induced cellular signatures (LINCS) library chemical screen In vivo model of inducible mutant KRAS-positive lung cancer. A well- established inducible mutant KRAS lung cancer mouse model has The Kinase Inhibitor Focused Library, LINCS, was utilized for a chemical 21 been described previously. Additional details are provided in the screen designed to identify selective kinase inhibitors capable of Supplementary Information. synergizing with the mTOR inhibitor, Torin1,16 against mutant NRAS- driven cells (see schematic, Figure 1a). The LINCS library is available from In vivo model of mutant KRAS-positive leukemia. NB4 cells were Harvard Medical School/NIH LINCS program (https://lincs.hms.harvard.edu/) transduced with a retrovirus encoding firefly luciferase (MSCV-Luc), and and contains 202 known selective and potent kinase inhibitors. selected with neomycin (NB4-luc þ ). Six-week-old female NSG mice (Jackson Laboratories, Bar Harbor, ME, USA) were administered 1 Â 106 þ Cell lines and cell culture NB4-luc cells via tail vein injection, followed 4 days later by imaging and quantification of total body bioluminescence as previously described.19 IL-3-dependent murine Ba/F3 cells were transduced with NRAS G12D-or Treatment cohorts with matched tumor burden were established and mice KRAS G12D-containing murine stem cell virus (MSCV) retroviruses were orally administered 1 Â daily 10 mg/kg MK-1775, 10 mg/kg Torin 2 or harboring an internal ribosome entry site-green fluorescent protein and a combination. Mice were treated on day 1 of dosing with 15 mg/kg Torin rendered growth factor-independent via IL-3 withdrawal from the culture 2 alone or in combination with 10 mg/kg MK-1775, and thereafter, because media (to develop Ba/F3-NRAS-G12D and Ba/F3-KRAS-G12D cells, respec- of drug tolerance concerns, the dose of Torin 2 was lowered to 10 mg/kg tively). Ba/F3-NRAS G12D myrAKT cells (expressing constitutively active, for 6 consecutive days before the final imaging. Only viable mice were myristoylated AKT) were created by transducing pMIG myrAKT to Ba/F3- censored for assessment of leukemia burden via serial bioluminescence NRAS-G12D-neo and sorting GFP-positive cells. imaging on days 3 and 7 of drug treatment. Studies were performed with The human mutant NRAS-expressing AML line, P31-FUJ, and mutant Animal Care and Use Committee protocols at Dana-Farber Cancer Institute. KRAS-expressing AML lines, SKM-1 (KRAS K117N), NOMO-1 (KRAS G13D) For both in vivo studies, MK-1775 was dissolved in a solution containing and NB4 (KRAS A18D), were obtained from Dr Gary Gilliland. Mutant NRAS- 0.5% methycellulose and 0.5% Tween 80 (Sigma, St Louis, MO, USA). Torin expressing ALL lines, PF-382 and NALM6, were obtained by Dr Thomas 2 was dissolved in 30% capsitol (Sigma). Look and Dr David Weinstock, respectively. The human AML-derived, wild-type (wt) RAS-expressing line, MOLM14,17 was provided by Dr Scott Armstrong. MOLM14 cells were transduced with the FUW-Luc-mCherry- RESULTS 18 puro lentivirus as previously described. The wt RAS-expressing line, Identification of Wee1 inhibitor, MK-1775, as able to potentiate the HEL, was purchased from American Type Culture Collection (Manassas, effects of mTOR inhibition against mutant NRAS-expressing cells VA, USA). Information about culturing conditions and solid tumor cell lines are Single-agent monotherapies targeting either the PI3K/mTOR or provided in the Supplementary Information. MEK/ERK pathway, the two most critical signaling cascades downstream of activated RAS, have shown only modest effects in pre-clinical in vivo studies and in human clinical trials. However, Chemical compounds and biologic reagents a number of drug combinations centered on MEK inhibition, A listing of chemical compounds and biologic reagents is shown in the including addition of PI3K/mTOR/AKT inhibitors or conventional Supplementary Information. chemotherapy agents, have led to promising results in multiple RAS-expressing malignancies. In contrast, effective treatment Proliferation studies, cell cycle analysis and apoptosis assay combinations focused on mTOR inhibition are limited up to date. Details are provided in the Supplementary Information. Taking advantage of the LINCS chemical library, we performed a synergy screen to search for drugs with a favorable pharmacological profile that could synergize with the mTOR inhibitor, Torin Antibodies and immunoblotting 1 (see schematic, Figure 1a). We carried out our initial screen in a A listing of antibodies is shown in the Supplementary Information. mutant NRAS-transformed Ba/F3 cell line with parental Ba/F3 as Protein lysate preparation and immunoblotting were carried out as 19 baseline control. As expected, both MEK (PD0325901) and PI3K previously described. (GSK2126458) inhibitors were observed to synergize with Torin 1 against mutant NRAS-expressing cells (see schematic, Figures 1a Drug combination studies and 2), partially validating this strategy. The Chou-Talalay method20 was employed for drug combination studies. Surprisingly, however, the screen also identified the Wee1 Additional details are provided in the Supplementary Information. inhibitor, MK-1775, which has not been previously shown to

Leukemia (2015) 27 – 37 & 2015 Macmillan Publishers Limited Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 29

Figure 1. Identification of Wee1 inhibitor, MK-1775, as able to potentiate the effects of mTOR inhibition against mutant NRAS-expressing cells via inhibition of AKT, 4E-binding protein 1 (4E-BP1) and s6 kinase (S6K) phosphorylation. (a) Schematic of LINCS library chemical screen. 384- well plates were seeded with either parental Ba/F3 cells cultured in the presence of 15% WEHI (as a source of IL-3), which were used as a control to eliminate inhibitors that exhibit off-target toxicity and interfere with IL-3-mediated signaling, or Ba/F3-NRAS- G12D cells (growth factor independent). Included in the screen were cell-containing plates that only received LINCS library drugs (300 nM, determined to be the optimal screening concentration) for the purpose of assessing single-agent activity of the LINCS library compounds, or plates that contained LINCS library compounds (300 nM) plus the mTOR inhibitor, Torin 1 (20 nM, which was determined to be close to the IC50 against mutant NRAS-expressing cells). Dimethyl sulfoxide (vehicle) wells and Torin 1-only wells were included as controls in the plates. Following pintool administration of the library compounds, the 384-well plates were incubated for 2 days before administration of Cell Titer Glo (Promega, Madison, WI, USA) and analysis of bioluminescence using a plate reader. Ideal candidates for further investigation were LINCS library compounds showing minimal activity as single agents against parental Ba/F3 cells, yet potentiation of the efficacy of Torin 1 against NRAS- driven cells. ‘Hits’ were validated for synergizing potential using several approaches, including cellular proliferation assays, signaling studies and in vivo analysis. (b–e) Approximately 2-day proliferation studies performed with MK-1775, combined with AZD8055 or WYE-125132 against parental Ba/F3 and Ba/F3-NRAS-G12D cells. (f–h) Effect of approximately 2-h treatment of Ba/F3 or Ba/F3-NRAS-G12D cells with MK-1775 (75 nM), alone and combined with mTOR inhibitor, AZD8055 (20 nM), on phosphorylation of 4E-BP1, AKT and S6K, as measured by immunoblotting. PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol (3,4,5)-triphosphate; TSC1 (hamartin), tuberous sclerosis protein 1; TSC2 (tuberin), tuberous sclerosis protein 2; WB, western blot; WEHI, WEHI-3 cells (myelomonocytic leukemia, macrophage- like, Balb/C mouse cells, available from ATCC: TIB 68). enhance anti-tumor effects of mTOR inhibition, despite prior The combination of MK-1775 and mTOR inhibition against evidence supporting its role as a synergistic partner for cytotoxic Ba/F3-NRAS-G12D cells correlates with inhibition of AKT, chemotherapy (see schematic, Figures 1a and 2). We further 4E-binding protein 1 (4E-BP1) and s6 kinase phosphorylation validated the observed synergy using additional selective mTOR mTOR inhibitors as single agents led to substantial G1 arrest in inhibitors, AZD8055, WYE125132 and Torin 2, and confirmed its Ba/F3-NRAS-G12D cells approximately 48 h after treatment, but activity against Ba/F3-NRAS-G12D cells, but not parental Ba/F3 not parental Ba/F3 cells (Supplementary Figure 1). However, cells (Figures 1b–e and 2). addition of MK-1775 did not result in significant differences in cell

& 2015 Macmillan Publishers Limited Leukemia (2015) 27 – 37 Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 30

Figure 2. Combination indices for proliferation studies testing MK-1775 alone and combined with targeted inhibitors of mutant RAS signaling. MK-1775 is abbreviated as ‘MK.’ Values less than 0.9 indicate synergy and are in shades of red (darker shades mean higher synergy). Values greater than 0.9 do not indicate synergy and are colored white. Mutant RAS-expressing Ba/F3 cells are shown in dark blue font. Parental Ba/F3 cells are shown in light blue font. Mutant NRAS-positive melanoma cell lines are shown in orange font. Mutant KRAS-positive colorectal cancer cell lines are shown in dark green font. Mutant KRAS-expressing pancreatic cancer cell lines are shown in light green font. Primary AML patient cells are shown in dark purple font.

cycle progression as compared with mTOR inhibitor only-treated the question whether combined Wee1 and mTOR inhibition also cells (mTOR inhibitor ¼ 20 nM, MK-1775 ¼ 75 nM; Supplementary leads to enhanced inhibitory effects in mutant KRAS-expressing Figure 1), suggesting that the role of Wee1 as a cell cycle entry cells. Using a Ba/F3 cell line dependent on the expression of checkpoint regulator may not have a significant contribution to a KRAS G12D mutant, we confirmed the synergistic effects the observed synergy. We detected enhanced suppression of between MK-1775 and mTOR inhibition (Figures 3a, b and 2). phosphorylation of signaling molecules downstream of mutant Consistent with the results in mutant NRAS-expressing Ba/F3 RAS within the PI3K/mTOR/AKT pathway, including 4E-BP1 and cells, we also observed decreased activity of phospho-4E-BP1, AKT, in mutant NRAS-expressing Ba/F3 cells with the combination phospho-AKT and phospho-s6 kinase in Ba/F3-KRAS-G12D cells of Wee1 and mTOR inhibition (Figures 1f and g). In contrast, no drug (Figures 3c–e). combination effects were observed in wild-type RAS-expressing parental Ba/F3 cells (Figures 1f and g). The drug combination also led to greater suppression of S6 kinase phosphorylation (Figure 1h), Combined Wee1 and mTOR inhibition induces apoptosis in although no further decrease in MAPK phosphorylation in mutant mutant RAS-expressing cells RAS-expressing cells, as compared with single-agent effects, was Importantly, consistent with the increased mTOR/AKT inhibition, observed (Supplementary Figure 2). These results suggest the the combination of MK-1775 and mTOR inhibition led to a MAPK pathway may have a less important role for the synergy of higher increase in apoptotic cell death than either agent alone the two drugs in this particular genetic setting. when tested against mutant NRAS- and KRAS-expressing cells (Figure 3f), confirming the observed synergy for anti-cancer effects. This finding is further supported by increased Combined Wee1 and mTOR inhibition is effective against phosphorylated histone H2A.X, a marker for DNA damage, Ba/F3-KRAS-G12D-expressing cells which is required for DNA fragmentation during apoptosis and Because of the critical function of the PI3K/mTOR/AKT pathway phosphorylated in response to apoptotic signals (Supplementary in the downstream signaling of multiple RAS isotypes, we asked Figure 3).

Leukemia (2015) 27 – 37 & 2015 Macmillan Publishers Limited Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 31

Figure 3. Combined Wee1 and TORC inhibition is effective against Ba/F3-KRAS-G12D-expressing cells and induces apoptosis in mutant NRAS- and mutant KRAS-expressing cells. (a, b) Approximately 2-day proliferation studies performed with MK-1775, combined with AZD8055 or WYE-125132 against Ba/F3-KRAS-G12D cells. (c–e) Effect of approximately 2-h treatment of Ba/F3-KRAS-G12D cells with MK-1775 (75 nM) alone and combined with mTOR inhibitor, AZD8055 (20 nM), on phosphorylation of 4E-BP1, AKT and S6K, as measured by immunoblotting. (f) Approximately 3-day treatment of Ba/F3, Ba/F3-NRAS-G12D and Ba/F3-KRAS-G12D cells, respectively, with MK-1775 (75 nM), AZD8055 (20 nM), WYE125132 (20 nM) or a combination of MK-1775 þ AZD8055 or MK-1775 þ WYE125132 before analysis of apoptotic death.

MK-1775 selectively potentiates the anti-leukemic effects of mTOR In order to confirm that Wee1 inhibition has a role in the inhibition on proliferation of human mutant KRAS-expressing observed drug combination-induced apoptosis and synergy acute leukemia cells through CDK1 activation, we tested the ability of the CDK1 We validated our findings from the Ba/F3 system in mutant inhibitor, R0-3306, to block Wee1 and mTOR inhibitor-induced KRAS-expressing human acute leukemia cell lines. As shown in apoptosis in mutant RAS-expressing cells. Co-treatment of NB4 cells Figures 4a–d, the combination of Wee1 and mTOR inhibition with MK-1775 þ Torin 2 and R0-3306 led to a partial rescue of cells was synergistic against human mutant KRAS-expressing AML from the proapoptotic effects of the drug combination (Figure 5d). cell lines, SKM-1, NOMO-1 and NB4. In addition, the drug Modest rescue of drug combination effects on cellular proliferation combination corresponded to a higher fraction of apoptotic was also observed with R0-3306 treatment (Supplementary cells and poly (ADP-ribose) polymerase cleavage than either Figure 4). Importantly, shRNA knockdown of Wee1 in mutant drug alone in mutant KRAS-expressing cells, as compared with KRAS-expressing NB4 cells led to an increased sensitivity of cells to wild-type RAS-expressing cells (Figures 4e and f). the anti-proliferative effects of MK-1775 and Torin 2, both as single agents and combined (Figures 5e, f). These results suggest that the synergy observed between MK-1775 and mTOR inhibition is at least Wee1 kinase is the main target of MK-1775 that mediates synergy in part due to targeting of Wee1 by MK-1775. with mTOR inhibitors To exclude potential off target effects of MK-1775 that may contribute to the observed synergy with mTOR inhibition, we first MK-1775 selectively potentiates the anti-leukemic effects of mTOR confirmed that MK-1775 potently decreased the levels of inhibition on proliferation of human mutant NRAS-expressing phosphorylated cdc2 (CDK1), a direct substrate of Wee1, in a acute leukemia cells concentration-dependent manner (Figure 5a) in Ba/F3-NRAS-G12D We next validated our findings from the Ba/F3 system in mutant cells that expressed higher protein levels of NRAS than parental NRAS-expressing human acute leukemia cell lines. Similar to the Ba/F3 cells (Figure 5b). Similarly, MK-1775 inhibited phosphoryla- Ba/F3 results, MK-1775 was observed to effectively combine, tion of CDK1 in human active KRAS-expressing NB4 cells respectively, with the selective mTOR inhibitor, WYE125132, (Figure 5c). against the mutant NRAS-expressing lines, PF-382 (ALL), NALM6

& 2015 Macmillan Publishers Limited Leukemia (2015) 27 – 37 Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 32

Figure 4. MK-1775 selectively potentiates the anti-leukemic effects of mTOR inhibition on proliferation of human mutant KRAS-expressing AML cells via induction of apoptosis. (a–c) Dose–response curves for mutant KRAS-expressing human AML cells (SKM-1, NOMO-1 and NB4) showing single agent versus drug combination effects following approximately 3 days of treatment. (d) Calcusyn combination indices derived from six-point concentration proliferation experiments. The cutoff for nearly additive effects (CI: 1.1) is marked by a dashed line. (e) Approximately 3-day treatment of HEL, NOMO-1 and NB4 cells, respectively, with MK-1775 (75 nM), Torin 2 (20 nM) or a combination of MK-1775 þ Torin 2 before analysis of apoptotic death. (f) Immunoblot showing effect of MK-1775 þ /-Torin 2 on poly (ADP-ribose) polymerase cleavage in HEL and NB4 cells.

(ALL) and P31-FUJ (AML; Figures 6a–c and f). In contrast, our results MK-1775 potentiates the effects of PI3K inhibition against mutant suggest potential antagonism between MK-1775 and WYE125132 RAS-positive cells against wt RAS-expressing lines, such as HEL or MOLM14 (Figures As the PI3K/AKT/mTOR pathway may be particularly important for 6d–f). Similar results were observed when MK-1775 was com- the observed synergy, we investigated whether MK-1775 was able bined, respectively, with mTOR inhibitors Torin 1, Torin 2 or to synergize with inhibitors targeting components within the AZD8055 (data not shown).We also tested the combined Wee1 PI3K/AKT/mTOR pathway other than targeted mTOR inhibitors. and mTOR inhibition in mutant NRAS-expressing primary AML The results suggest that MK-1775 selectively synergized patient cells, and observed more cell death with the combination with ZSTK474, an inhibitor of PI3K isoforms, against mutant than either agent alone (Figures 6g and h, Supplementary Figure 5), RAS-expressing Ba/F3 cells, however, this drug combination was with combination indices suggestive of synergy (Figure 2). These antagonistic against parental Ba/F3 cells (Supplementary Figure 7). data support the potential clinical utility of this drug combination for mutant NRAS-positive AML. MK-1775 potentiates the effects of mTOR inhibition against AKT partially mediates potentiation of mTOR inhibition by mutant RAS-expressing solid tumors MK-1775 Having established the effectiveness of MK-1775 combined with To assess the signiﬁcance of AKT in the observed synergy between mTOR inhibition against mutant RAS-positive acute leukemia, we MK-1775 and mTOR inhibitors, we compared the effects of drug investigated whether this particular targeting strategy is also treatment on Ba/F3-NRAS-G12D cells and Ba/F3-NRAS-G12D cells efﬁcacious against other mutant RAS-positive malignancies. The expressing constitutively active, myristoylated AKT (Ba/F3-NRAS- incidence of NRAS mutations in human melanoma has been G12D-AKT (myr)). Whereas the activity of MK-1775 was not reported to be as high as 28%,22 whereas the incidence of KRAS substantially different between the two cell lines, Ba/F3-NRAS- mutations ranges between 20 and 40% in colorectal cancer,23 75 G12D-AKT (myr) cells were less sensitive to the effects of mTOR and 95% in pancreatic cancer24 and 15 and 30% in lung inhibition (Supplementary Figure 6). There was a modest right- adenocarcinoma.25 Both AZD8055 and WYE125132, respectively, ward shift in the drug combination dose-response curve in Ba/F3- synergized with MK-1775 against mutant NRAS-positive NRAS-G12D-AKT (myr) cells, as compared with Ba/F3-NRAS-G12D melanoma cell lines, K023, K033 and M13 (Supplementary cells, as well as corresponding higher combination indices Figure 8 and Figure 2). In addition, comparable potentiating (Supplementary Figure 6), suggesting that AKT may at least in effects of MK-1775 for mTOR inhibitors were also observed in part contribute to the observed synergy between MK-1775 and three mutant KRAS-expressing colorectal cancer cell lines, HCT116, mTOR inhibition. DLD1 and SW480 (Supplementary Figure 9 and Figure 2), and in

Leukemia (2015) 27 – 37 & 2015 Macmillan Publishers Limited Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 33

Figure 5. Wee1 kinase is the main target of MK-1775 that mediates synergy with TORC inhibitors. (a, c) Effect of approximately 2 h treatment of Ba/F3-NRAS-G12D cells or NB4 cells with MK-1775 on phosphorylation of cdc2 (CDK1), as measured by immunoblotting. (b) NRAS expression in Ba/F3 versus Ba/F3-NRAS-G12D cells. (d) Measurement of apoptosis following approximately 48 h treatment of NB4 cells with MK-1775, Torin 2, MK-1775 þ Torin 2 or MK-1775 þ Torin 2 in the presence of a CDK1 inhibitor at the indicated concentrations. (e) Proliferation of approximately 48 h MK-1775 (75 nM)- and Torin 2 (20 nM)-treated NB4 cells following knockdown of Wee1 by shRNA (hp2) as compared with GFP control. (f) Validation of Wee1 KD efﬁciency in NB4 cells. Combo, combination; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

two mutant KRAS-expressing pancreatic cancer cell lines, PATO P6 murine model. We used a well-established genetically engineered and MIA PaCa (Supplementary Figure 10 and Figure 2). MK-1775 mouse lung cancer model that harbors an activating kras also enhanced the effects of mTOR inhibition against active KRAS- mutation (G12D).21 Specifically, mice harboring conditional expressing human lung cancer lines, A549, H1355, H441, H1944 activating mutation (G12D) at the endogenous kras locus were and H1792 (Figure 7a and Supplementary Figure 11). Finally, MK- activated by nasal instillation of 5 Â 106 p.f.u. of adenovirus- 1775 synergized with Torin 1 or Torin 2 against p53-kd/KRAS- and encoding cre recombinase. Expression of cre recombinase in Lkb-1/p53-kd/KRAS-expressing murine lung cancer cell lines infected lung epithelial cells removes loxp-flanked stop cassette derived from in vivo models (Supplementary Figure 12, located before ATG start codon of the mutant Kras gene Supplementary Table 1). (Supplementary Figure 13). Induced kras mice express activating kras G12D mutant in lung epithelial cells, and have a disease latency of 13–15 weeks after induction. Combination of MK-1775 and Torin 2 is effective against mutant With this model, we were able to detect significant tumor KRAS-positive lung cancer and mutant KRAS-positive leukemia in regression when tumor-bearing mice were treated by the mouse models MK-1775 and Torin 2 combination (Figures 7b and c). Progressive Having demonstrated the ability of MK-1775 to potentiate the disease is defined as 20% progression in tumor volume over the efficacy of mTOR inhibition against mutant NRAS-positive baseline scan performed before treatment starts. Partial response leukemia and melanoma in vitro, and given the observed synergy is defined as 30% or more regression in tumor volume compared between Wee1 inhibition and mTOR inhibition against mutant with baseline. Stable disease is between 20% progression and 30% KRAS-positive colorectal, pancreatic and lung cancer in vitro,we regression. The combination therapy achieved a nearly 81% tested this combination against KRAS-positive lung cancer in a disease control rate (43% partial response and 28% stable

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Figure 6. MK-1775 selectively potentiates the anti-leukemic effects of mTOR inhibition on proliferation of human mutant (Mut) NRAS- expressing acute leukemia cells. (a–e) Dose–response curves for Mut NRAS-expressing acute leukemia cells (PF-382, NALM6, P31-FUJ) or wt RAS-expressing acute leukemia cells (HEL, MOLM14) showing single agent (MK-1775 or WYE125132) versus drug combination effects following approximately 2 days of treatment. (f) Calcusyn combination indices derived from six-point concentration proliferation experiments. The cutoff for nearly additive effects (CI: 1.1) is marked by a dashed line. (g, h) Mut NRAS-positive AML patient samples treated with MK-1775, WYE125132 or a combination of both.

disease). In contrast, single-agent treatment with either MK-1775 value in inhibiting the progression of mutant RAS-driven or Torin 2 showed 100% and 80% progressive disease, respectively malignancies. (Figures 7b and c). Increased disease suppression by the Wee1 and mTOR inhibitor combination, as compared with single agents, was similarly DISCUSSION observed in a murine model of mutant KRAS-positive AML. The Mediation of the effects of RAS by prominent signaling pathways combination of MK-1775 and Torin 2 was more effective at such as PI3K//PTEN/AKT/mTOR and Raf/MEK/ERK has prompted suppressing the growth of NB4-luc þ cells than either drug the testing of targeted inhibitors of these pathways as a strategy alone following 1 week of drug administration (Figures 7d–f). The to treat mutant RAS-driven malignancies. Unfortunately, inhibition comparable trend observed for the drug combination versus of PI3K or mTOR alone has limited efﬁciency (reviewed in Ward single-agent effects in the mutant RAS leukemia model et al.26) and has further contributed to the clinical sense that supports results observed in the mutant RAS lung cancer model. targeting RAS in general has been disappointing.27,28 In part, the Taken together, these results suggest that the combination of a limited efﬁcacy of inhibitors of Raf/MEK/Erk signaling or PI3K/Akt Wee1 inhibitor and an mTOR inhibitor may be of potential clinical in mutant RAS-positive cancer is believed to be due to negative

Leukemia (2015) 27 – 37 & 2015 Macmillan Publishers Limited Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 35

Figure 7. In vivo investigation of the combined effects of Wee1 and mTOR inhibition in murine models of active KRAS. (a) Calcusyn combination indices derived from six-point concentration proliferation experiments, investigating the combination of MK-1775 and mTOR inhibitors against human active KRAS-expressing lung cancer cell lines. The cutoff for nearly additive effects (CI: 1.1) is marked by a dashed line. (b) In vivo effects of Torin 2 (20 mg/kg 1 Â daily), MK-1775 (10 mg/kg 1 Â daily) or a combination of the two agents using an inducible active KRAS model of lung cancer. Each column represents tumor volume changes after 2 weeks of indicated treatment for an individual mouse by comparing total lung tumor volume before and after treatment. Negative and positive percentage indicates tumor regression and progression, respectively. A two-tailed t-test was used to calculate the P-value between treatment groups. PD, progressive disease; PR, partial response; SD, stable disease. (c) Representative MRI scans detecting tumor burden changes before and after indicated treatments. Arrows, tumors; H, Heart. (d–f) In vivo effects of Torin 2, MK-1775 or a combination of the two agents using a murine model of mutant KRAS-positive leukemia. (d) Mouse bioluminescence images taken before treatment (baseline) and after 1 week of drug treatment of NSG mice harboring NB4-luc þ cells. (e) Bioluminescence values plotted over time for single agent- and combination-treated mice. (f) Fold leukemia induction plotted over time for single agent- and combination-treated mice. Combination-treated mice were tested in a pilot titration study testing for Torin 2 tolerance/toxicity in which they received 0, 5 or 10 mg/kg Torin 2 1 Â daily for 1 week before tail vein-injection of NB4-luc þ cells. Drug administration was terminated several days before cell inoculation and observed to have no inﬂuence on starting leukemia burden. feedback loops and compensatory activation of bypass signaling drug-resistant glioma and other cancers.29,30 Also included pathways, leading the many current efforts to simultaneously was WYE125132 (Wyeth (Pﬁzer)), which shows 5000-fold higher target multiple effectors in mutant RAS-positive cancers. selectivity for mTOR over PI3K and has demonstrated anti-cancer In the present study, we introduce a novel chemical screen activity against solid tumors, including gliomas and cancers of the approach, using the LINCS chemical library, to identify agents with kidney, breast and lung.31 Finally, we used Torin 2,32 asecond- activity in the presence of mTOR inhibition in mutant RAS- generation ATP-competitive inhibitor that was developed by expressing cells. LINCS library compounds were anticipated to be optimizing the earlier generation inhibitor, Torin1.8 Torin 2 potently useful because of their limited spectrum of kinase targets, inhibits the signaling complex, mammalian target of rapamycin therefore giving the potential of more successfully identifying complex 1 (mTORC1), with around 800-fold selectivity for cellular real target kinases. Consistent with what has been reported in the mTOR versus PI3K, and potently inhibits additional targets including literature, mTOR inhibition was potentiated by inhibitors of the PI3K-like kinase family kinases (including ATM, DNA-PK and ATR).33 PI3K/AKT and MEK derived from the screened chemical library. mTORC1 promotes protein synthesis by phosphorylation of We also surprisingly observed enhancement of the effects of 4E-BP1 and p70S6K, whereas mammalian target of rapamycin mTOR inhibition by the targeted Wee1 inhibitor, MK-1775. The complex 2 (mTORC2) promotes survival and migration through notion that the relevant target of MK-1775 was indeed Wee1 was phosphorylation of AKT and activation of RhoGTPase (reviewed in supported by initial studies with an inhibitor relevant to Wee1 Bracho-Valdes et al.34). In the present study, the strongest signaling, as well as by knockdown of Wee1 expression. suppression of phosphorylation of AKT and 4E-BP1 was Our studies used several well-characterized mTOR inhibitors, observed by the combination of MK-1775 and mTOR inhibition including AZD8055, a pan-mTOR inhibitor developed by AstraZeneca, in mutant RAS-expressing cells, suggesting MK-1775 potentiation which is being investigated in a clinical trial (NCT01316809) aimed at of mTOR inhibitor effects in this system.

& 2015 Macmillan Publishers Limited Leukemia (2015) 27 – 37 Wee1: novel target for mutant RAS-positive cancer E Weisberg et al 36 Both 4E-BP1 and AKT have significant roles in apoptosis, and and limited tolerance.48 There is thus a need for more efficacious loss of their activity correlates with the increased apoptosis and safer therapeutic strategies, and combination therapy based observed in mutant RAS-expressing cells by the MK-1775 þ mTOR on small molecule, targeted pharmacological inhibitors offers a inhibitor combination. Specifically, as activated AKT phosphor- promising alternative. ylates and inhibits pro-apoptotic Bcl-2 family members,35 the Here, we have focused primarily on the effects of Wee1 and increased dephosphorylation of AKT observed with MK- mTOR inhibition, combined, against mutant RAS-driven acute 1775 þ mTOR inhibition supports a possible role for AKT in leukemia. Our findings additionally extend to a variety of solid enhanced induction of apoptosis in mutant RAS-positive cells. tumors characterized as harboring RAS mutations. High expression Inhibition of mTOR signaling and dephosphorylation of 4E-BP1 of Wee1 was reported in malignant melanoma and was found to precede apoptosis, and in apoptotic cells, caspase-dependent correlate with poor disease-free survival, and abnormal Wee1 cleavage of 4E-BP1 leads to inhibition of its phosphorylation and expression was found in lung carcinoma.49,50 These findings an increase in its association with eukaryotic initiation factor 4E.36 suggest that there is potential clinical benefit to be gained from The increased dephosphorylation of 4E-BP1 observed with MK- using a Wee1 inhibitor as part of a treatment strategy against 1775 þ mTOR inhibition may similarly correlate with a possible these diseases. Importantly, however, malignancies driven by or role for 4E-BP1 in increased apoptosis in mutant RAS-expressing characterized by mutated RAS expression represent especially cells. challenging disease targets because of the difficulties associated It is unknown which malignancies will respond to or show with directly targeting the RAS protein, as well as feedback loops resistance to the pan mTOR inhibitors that are currently in clinical and the redundancies and complexities of RAS signaling that trials. For instance, KRAS was identified in one study as being a counteract effective targeting of signaling factors downstream significant genetic marker for resistance to PP-242, an inhibitor of of RAS. both mTORC1 and mTORC2 that is presently in early-stage clinical Our findings support the notion that an inhibitor of Wee1 may trials.37 The degree of effectiveness of mTOR inhibition in that be effectively combined with a targeted inhibitor of mutant RAS study correlated with the degree of inhibition of phosphorylation signaling against malignancies characterized by RAS mutations. of 4E-BP1. RAS activation also leads to hyper-activation of the This novel combination strategy thus warrants further investiga- serine/threonine kinase, AKT and increased AKT activation may tion as a therapeutic approach that could potentially override account for resistance to therapy.38 The complete inhibition of drug resistance associated with mTOR inhibition and that there- phosphorylation of 4E-BP1 and AKT by the combination of MK- fore may be of potential clinical benefit in mutant RAS-driven 1775 and mTOR inhibition, versus only partial inhibition of these malignancies. molecules by mTOR inhibition alone, points toward a potentially effective strategy to override insensitivity of mutant RAS- expressing cells to mTOR inhibition. CONFLICT OF INTEREST Importantly, cdc2 (CDK1), a direct substrate of Wee1 that is JDG receives research support and has a financial interest with Novartis Pharma AG. inhibited by Wee1, has been described as a proapoptotic The remaining authors declare no conflict of interest. mediator.39 Specifically, increased CDK1 activity has been observed to correlate with apoptosis and studies involving CDK1 inhibition have suggested that this increased activity may be ACKNOWLEDGEMENTS 39 significant for cell death. In the present study, we have shown We wish to acknowledge Catherine Sypher, Carmen Da Silva, Elsy Moreno, Lia that MK-1775, which inhibits Wee1, leads to dephosphorylation of Palazzolo and Daisy Moreno for their assistance with in vivo studies. NG is supported CDK1 and presumed activation of CDK1. Co-treatment of mutant by NIH LINCS grant HG006097. MS is supported by NIH grant CA134660. 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